The molecular pathogenesis of fully virulent, wild-type Y. pestis in relevant animal models has been relatively neglected because of the scarcity of secure BSL-3 facilities and trained personnel certified to work with this Class A select agent. The threat of bioterrorism and the emergence of multiply-antibiotic resistant strains of Y. pestis increases the urgency for a more detailed understanding of the host-pathogen relationship at the molecular level that may lead to the design of improved medical countermeasures and diagnostics. We have established mouse and rat models of bubonic plague that incorporate flea-to-rodent transmission to investigate the role of specific Y. pestis virulence factors and to characterize the host response to naturally acquired infection. We have characterized the kinetics, microbiology, and histopathology of bubonic plague in rats following intradermal injection of Y. pestis, and used this model to characterize the gene expression profile of Yersinia pestis in the infected lymph node during bubonic plague using whole-genome microarray technology. Based on these results, we tested the virulence of specific Y. pestis mutant strains to determine the role of bacterial genes predicted to be important in resistance to the host innate immune response. During the past year, we determined that Ail, a Y. pestis outer surface protein, is an essential virulence factor and identified the reason: Ail is required to prevent a protective response by polymorphonuclear leukocytes (also called PMNs or neutrophils), a phagocytic cell that is an important innate defense against infection. We are conducting trials to determine the efficacy of the Ail protein as a component of a third-generation anti-plague vaccine. In addition to Ail, we are studying other Y. pestis factors that counteract PMN function, in particular the Y. pestis insecticidal-like toxins that are upregulated in the flea. We now have several lines of evidence that the PMN response correlates with successful outcome to infection, and this aspect of host-pathogen interaction has become a focus of our lab. We have developed models, including in vivo imaging techniques, to examine the initial encounter of Y. pestis with the host innate immune response, particularly PMNs, in the dermis after transmission by flea bite. In collaboration with Dr. Susan Buchanan (NIDDK), we have evaluated the efficacy of a novel hybrid lysin protein as a therapeutic agent for plague.